This application describes the candidate's research on the genetics of red blood cell development (erythropoiesis) to be performed within the context of a 5-year mentored career development plan. His ultimate goal is to become an independent physician-scientist in the area of laboratory-based, academic hematology- oncology. Under the guidance of his primary research mentor Dr. Gerd Blobel at the Children's Hospital of Philadelphia (CHOP), and his co-mentor Dr. Mitchell Weiss at St. Jude Children's Research Hospital, Dr. Paralkar has developed a structured training plan consisting of intensive laboratory research, coursework in RNA biology and bioinformatics, regular participation at scientific conferences, and oversight by an experienced faculty advisory committee. Drs. Blobel and Weiss have extensive experience in training scientists and physician-scientists, and the educational/training environments at CHOP and the University of Pennsylvania are outstanding. Dr. Paralkar's laboratory work centers on long non-coding RNAs (LncRNAs), a recently appreciated class of genetic material that modulates gene expression and regulates diverse aspects of normal and pathological mammalian development. Thousands of LncRNAs are believed to exist, but only few have been studied in detail. Dr. Paralkar has identified a novel LncRNA named Drip27 that regulates the critical cell cycle inhibitor p27. This proposal investigates the hypothesis that Drip27 LncRNA directly activates p27 transcription, and thereby regulates cell division and replication during erythropoiesis. The long term goal of this research is to define the role of Drip27 in normal hematopoietic development and associated diseases such as blood cancers. In preliminary studies, Dr. Paralkar used comprehensive RNA-Sequencing and bioinformatic tools to identify hundreds of novel LncRNAs in mouse and human erythroid precursors. He chose a novel conserved LncRNA named `Drip27,' and he used CRISPR technology to delete it in an erythroid cell line. Deletion led to a significant reduction i the expression of the crucial cell cycle inhibitor p27. This proposal aims to study the effect of Drip27 transcript loss on erythropoiesis by generating mouse ES cells with truncated Drip27 transcript and differentiating them into erythroblasts (Aim 1). The mechanisms by which Drip27 functions will be examined by imaging the subcellular localization of Drip27 RNA molecules and by assessing for the presence of chromosomal looping between the Drip27 and the p27 loci (Aim 2). Published work demonstrates that many LncRNAs regulate gene expression through physical interactions with transcription factors/co-factors/chromatin modifying complexes that orchestrate global gene expression. To further define the mechanism by which Drip27 acts on the p27 locus, proteins binding to Drip27 will be identified (Aim 3). These three aims, put together, will establish the role played by Drip27 in erythropoiesis and the mechanism by which it regulates its target gene. If successful, this work will illuminate novel regulatory mechanisms in erythropoiesis and paradigms for noncoding RNA function. In turn, these findings are likely to produce insights into various blood diseases including anemias, myeloproliferative disorders and myelodysplastic syndromes. Performing this research in the context of a rigorous formal training plan within a highly supportive academic enviroment will provide the candidate essential data and professional tools for advancement into an independent position as a research oriented academic physician-scientist.